The long-term goal of this project is to understand the role(s) of the miRNA processing enzyme Dicer and of microRNA (miRNA) molecules in the regulation of mammalian adipocyte differentiation and fat metabolism. miRNAs are small ~22 nucleotide non-coding ribonucleic acids that regulate gene expression by interacting with target mRNA transcripts. Little is known about the precise biological roles of most mammalian miRNAs, but the abundance of different miRNA molecules encoded in mammalian genomes and their specific spatial and temporal patterns of expression suggest that these molecules may regulate a broad array of functions, including development, cell growth, and cell differentiation. Several recent publications indicate potential roles for miRNA molecules in adipogenesis in Drosophila, and in adipocyte differentiation and in glucose-dependent insulin exocytosis in mammalian cultured cells. These findings suggest that miRNA molecules regulate mammalian fat metabolism in vivo and may be therapeutic targets for treatment of diabetes mellitus. Dicer is a key enzyme in the processing of miRNA molecules. Our lab has recently utilized gene targeting in ES cells to create a Dicer-conditional allele in mice. The use of this new model circumvents the early embryonic lethal phenotype of Dicer-null mice, permitting analysis of the roles of Dicer and, by extension, miRNA in a wide variety of biological events. We have used viral-mediated transduction of Cre recombinase into mouse embryonic fibroblasts derived from these Dicer conditional mice to generate primary fibroblasts lacking Dicer activity. We propose to utilize these viable, Dicer-null, primary cells to study the effects of miRNA on adipocyte differentiation in vitro. Furthermore, we propose to cross the Dicer conditional mice with Ins2-Cre transgenic mice in order to analyze insulin secretion and adipogenesis in mice that lack Dicer in pancreatic beta cells. These experiments should provide definitive in vivo evidence for RNAi - mediated regulation of insulin production and mammalian fat metabolism. Relevance to Public Health: Diabetes and other obesity-related diseases affect a significant percentage of the population. By identifying the role of Dicer and of miRNA molecules in fat metabolism, these experiments will determine if these molecules are attractive targets for therapeutic intervention in diabetes and obesity.